Metal nanocluster beacons for detection of epigentic modifications

ABSTRACT

In one embodiment, compositions and methods are provided for the detection and/or quantification of epigenetic modifications in DNA. In particular aspects, probes are provided comprising fluorescent metal nanocluster beacons, which can selectively detect nucleic acids including an epigenetic modification.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/202,424, filed Aug. 7, 2015, the entirety of which isincorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“UTSBP1074US_ST25.txt”, which is 8 KB (as measured in MicrosoftWindows®) and was created on Aug. 5, 2016, is filed herewith byelectronic submission and is incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of molecularbiology. More particularly, it concerns nucleic acid based probes andtheir uses for detecting epigenetic modifications in nucleic acid.

2. Description of Related Art

Epigenetic modifications are regarded as fundamental elements in geneexpression regulation. DNA methylation, one such modification, playscrucial roles in widespread biological phenomena including host defensein bacteria and cell cycle regulation, gene imprinting, embryonicdevelopment and X-chromosome inactivation in mammals. Aberrant DNAmethylation patterns in gene promoters are closely associated withperturbations in gene expression and have recently been indicated as aleading cause of human cancers. Likewise, N⁶-methyladenosine, aubiquitous modification in prokaryotic and eukaryotic genomes, isrelated to many biological functions and human diseases in differenttissues and cancer cell lines with the highest levels detected in thebrain, heart and kidney (Meyer at al., 2014). Epigenetic modifications,such as N⁶-methyladenosine modification, are also involved in variousphysiological processes such as obesity, synaptic signaling, spermdevelopment, stem cell differentiation, circadian periods, yeastmeiosis, plant development and oogenesis. However, despite theimportance of epigenetic modifications and rapid and significantadvances in gene sequencing technology, methods for detecting andquantifying epigenetic changes remain elusive.

SUMMARY OF THE INVENTION

Embodiments of the present invention provide methods and compositionsfor the detection and quantification of epigenetic modifications innucleic acid sequences. For example, in a first embodiment there isprovided metal nanocluster beacon probes that can discriminate betweenunmodified and epigenetically modified nucleic acid sequences. In oneaspect, a probe set is provided comprising: (a) a first probe comprising(i) a hybridization sequence that is complementary to a first sequencein a test nucleic acid sample; (ii) a recognition nucleotide thatinteracts with a candidate nucleotide in the test nucleic acid sample,said candidate nucleotide being a candidate for the epigeneticmodification; and (iii) a nucleation sequence comprising a fluorescentmetal nanocluster; and (b) a second probe comprising (iv) an enhancersequence that enhances fluorescence of the fluorescent metal nanoclusterwhen the enhancer sequence interacts with the nucleation sequence of thefirst probe; (v) an abasic site; and (vi) a hybridization sequence thatis complementary to a second sequence in a test nucleic acid sample.Alternatively, a probe set of the embodiments may comprise: (a) a firstprobe comprising (i) a hybridization sequence that is complementary to afirst sequence in a test nucleic acid sample; (ii) an abasic site; and(iii) a nucleation sequence comprising a fluorescent metal nanocluster;and (b) a second probe comprising (iv) an enhancer sequence thatenhances fluorescence of the fluorescent metal nanocluster when theenhancer sequence interacts with the nucleation sequence of the firstprobe; (v) a recognition nucleotide that interacts with a candidatenucleotide in the test nucleic acid sample, said candidate nucleotidebeing a candidate for the epigenetic modification; and (vi) ahybridization sequence that is complementary to a second sequence in atest nucleic acid sample.

In a further embodiment there is provided provide a method for detectingan epigenetic modification in a test nucleic acid sample comprising: (I)hybridizing the test nucleic acid sample to a first and second probe,wherein (a) the first probe comprises (i) a hybridization sequence thatis complementary to a first sequence in the test nucleic acid sample;(ii) a recognition nucleotide that interacts with a candidate nucleotidein the test nucleic acid sample, said candidate nucleotide being acandidate for the epigenetic modification; and (iii) a nucleationsequence comprising a fluorescent metal nanocluster; and (b) the secondprobe comprises (iv) an enhancer sequence that enhances fluorescence ofthe fluorescent metal nanocluster when the enhancer sequence interactswith the nucleation sequence of the first probe; (v) an abasic site; and(vi) a hybridization sequence that is complementary to a second sequencein the test nucleic acid sample; (II) exposing the test nucleic acidsample and the first and second probes to an excitation light; and (III)detecting a fluorescence signal from the fluorescent metal nanocluster,thereby detecting an epigenetic modification in the test nucleic acidsample. In certain aspects, the recognition nucleotide is a guanine,cytosine, adenine, thymine or uracil. In specific aspects, therecognition nucleotide is a guanine or a thymine.

In still a further embodiment a method for detecting an epigeneticmodification in a test nucleic acid sample comprising: (I) hybridizingthe test nucleic acid sample to a first and second probe, wherein (a)the first probe comprises (i) a hybridization sequence that iscomplementary to a first sequence in the test nucleic acid sample; (ii)an abasic site; and (iii) a nucleation sequence comprising a fluorescentmetal nanocluster; and (b) the second probe comprises (iv) an enhancersequence that enhances fluorescence of the fluorescent metal nanoclusterwhen the enhancer sequence interacts with the nucleation sequence of thefirst probe; (v) a recognition nucleotide that interacts with acandidate nucleotide in the test nucleic acid sample, said candidatenucleotide being a candidate for the epigenetic modification; and (vi) ahybridization sequence that is complementary to a second sequence in thetest nucleic acid sample; (II) exposing the test nucleic acid sample andthe first and second probes to an excitation light; and (III) detectinga fluorescence signal from the fluorescent metal nanocluster, therebydetecting an epigenetic modification in the test nucleic acid sample. Incertain aspects, the recognition nucleotide is a guanine, cytosine,adenine, thymine or uracil. In specific aspects, the recognitionnucleotide is a guanine or a thymine.

In further aspects, the first sequence in a nucleic acid sample (such asa test or control sample) is positioned 5′ relative to the secondsequence of the nucleic acid sample. In certain aspects, the firstsequence in a nucleic acid sample (such as a test or control sample) ispositioned 3′ relative to the second sequence of the nucleic acidsample. In particular aspects, the first sequence in the nucleic acidsample and the second sequence of the nucleic acid sample are separatedby one nucleotide, which is the candidate nucleotide. Preferably, thefirst and/or second probe is at least 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29 or 30 nucleotides in length. In certain aspects, thefirst and/or second probe is a DNA or RNA probe. In further aspects, afirst and/or second probe of the embodiments is a chimeric DNA/RNAprobe.

In still further aspects, a first and second probe of the embodimentsare comprised on single nucleic acid molecule. For example, a singlenucleic acid molecule comprising a first and second probe may comprise,from 3′ to 5′ (i) a hybridization sequence that is complementary to afirst sequence in the test nucleic acid sample; (ii) a recognitionnucleotide that interacts with a candidate nucleotide in the testnucleic acid sample, said nucleotide being a candidate for theepigenetic modification; (iii) a nucleation sequence comprising afluorescent metal nanocluster; (iv) a loop sequence; (v) an enhancersequence that enhances fluorescence of the fluorescent metal nanoclusterwhen the enhancer sequence interacts with the nucleation sequence of thefirst probe; (vi) an abasic site; and (vii) a hybridization sequencethat is complementary to a second sequence in the test nucleic acidsample.

In further aspects, the step of detecting the fluorescence signal fromthe fluorescent metal nanocluster further comprises detecting a changein the fluorescence signal detected in the test nucleic acid sample ascompared to a control nucleic acid sample. For example, the change inthe fluorescence signal is a change in the wavelength of thefluorescence signal. In certain aspects, the change in the fluorescencesignal is a change of at least 1, 2, 3, 4, 5, 6, 7, 8 9, or 10 nm in thewavelength of the fluorescence signal. Thus, in some aspects thefluorescent metal nanocluster fluoresces in a visible color uponexcitation. In certain aspects, the excitation light is ultravioletlight, visible light, or near infrared light. For example, theexcitation light comprises a wavelength of from 200 nm to 2000 nm. Incertain aspects, the excitation light is provided by a laser.

In still further aspects, the step of detecting the fluorescence signalfrom the fluorescent metal nanocluster further comprises quantifying thefluorescence signal from the fluorescent metal nanocluster, therebyquantifying the proportion of nucleic acid molecules in the sample thatcomprise the epigenetic modification. For example, quantifying thefluorescence signal from the fluorescent metal nanocluster comprisesquantifying a change in the fluorescence signal detected in the testnucleic acid sample as compared to a control nucleic acid sample.

In certain aspects, a control nucleic acid sample comprises nucleic acidmolecules that are known to include or not include a given epigeneticmodification. In further aspects, the control nucleic acid samplecomprises nucleic acid molecules a known proportion of which include theepigenetic modification.

In certain aspects, an epigenetic modification for detection accordingto the embodiments is selected from the group consisting of5-methylcytosine, N⁴-methylcytosine, 5-hydroxymethylcytosine,N⁷-methylguanosine and N⁶-methyladenine. In exemplary aspects, theepigenetic modification is N⁶-methyladenine. In some aspects, the testand/or control nucleic acid sample is a DNA sample. In other aspects,the test and/or control nucleic acid sample is a RNA sample.

In certain specific aspects, a test nucleic acid sample comprises a GATnucleic acid sequence wherein the A position in the GAT sequence is thecandidate site for the epigenetic modification (e.g., a candidateN⁶-methyladenine position). In further aspects, the test nucleic acidsample comprises a GATC nucleic acid sequence wherein the A position inthe GATC sequence is the candidate site for the epigenetic modification.In these aspects, the recognition nucleotide for the probe is preferablya guanine.

In further aspects, the test nucleic acid sample comprises a CAG nucleicacid sequence, wherein the A position in the CAG sequence is thecandidate site for the epigenetic modification (e.g., a candidateN⁶-methyladenine position). In even further aspects, the test nucleicacid sample comprises a CTGCAG nucleic acid sequence, wherein the Aposition in the CTGCAG sequence is the candidate site for the epigeneticmodification. In these aspects, the recognition nucleotide for the probeis preferably a thymine.

In particular embodiments, the metal of the fluorescent metalnanocluster comprises silver, gold or copper. In exemplary methods, themetal of the fluorescent metal nanocluster comprises silver. Methods forthe use of such fluorescent metal nanoclusters are provided, forinstance, in U.S. Patent Publication No. 20140349289, which isincorporated herein by reference.

In some aspects, an enhancer sequence of the embodiments enhancesfluorescence emission of the fluorescent metal nanocluster by at least1.5, 2, 2.5, 3, 4 or 5-fold, when the enhancer sequence interacts withthe nucleation sequence of the first probe (e.g., when the enhancersequence is positioned in close proximity to the fluorescent metalnanocluster). In certain aspects, the enhancer sequence of the secondprobe comprises at least 30%, 35%, 40%, 45%, or 50% guanine positions.Preferably, the hybridization sequence of the first and/or second probeis at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20nucleotides in length.

In another embodiment there is provided a composition comprising (a) atest nucleic acid comprising a first and a second sequence, wherein saidfirst and second sequence are separated by one nucleotide, whichcomprises an epigenetic modification; (b) a first probe hybridized tothe test nucleic acid, the first probe comprising (i) a hybridizationsequence that is complementary to the first sequence in the test nucleicacid; (ii) a recognition nucleotide that interacts with the nucleotidethat comprises the epigenetic modification in the test nucleic acid; and(iii) a nucleation sequence comprising a fluorescent metal nanocluster;and (c) a second probe hybridized to the test nucleic acid, the secondprobe comprising (iv) an enhancer sequence that enhances fluorescence ofthe fluorescent metal nanocluster when the enhancer sequence interactswith the nucleation sequence of the first probe; (v) an abasic site; and(vi) a hybridization sequence that is complementary to a second sequencein the test nucleic acid sample.

In a further embodiment there is provided a composition comprising (a) atest nucleic acid comprising a first and a second sequence, wherein saidfirst and second sequence are separated by one nucleotide, whichcomprises an epigenetic modification; (b) a first probe hybridized tothe test nucleic acid, the first probe comprising (i) a hybridizationsequence that is complementary to the first sequence in the test nucleicacid; (ii) an abasic site; and (iii) a nucleation sequence comprising afluorescent metal nanocluster; and (c) a second probe hybridized to thetest nucleic acid, the second probe comprising (iv) an enhancer sequencethat enhances fluorescence of the fluorescent metal nanocluster when theenhancer sequence interacts with the nucleation sequence of the firstprobe; (v) a recognition nucleotide that interacts with the nucleotidethat comprises the epigenetic modification in the test nucleic acid; and(vi) a hybridization sequence that is complementary to a second sequencein the test nucleic acid sample.

Another embodiment concerns a kit comprising: (a) a control nucleic acidcomprising a first and a second sequence, wherein said first and secondsequence are separated by one nucleotide, which comprises an epigeneticmodification; (b) a first probe hybridized to the test nucleic acid, thefirst probe comprising (i) a hybridization sequence that iscomplementary to the first sequence in the control nucleic acid; (ii) arecognition nucleotide that interacts with the nucleotide that comprisesthe epigenetic modification in the control nucleic acid; and (iii) anucleation sequence comprising a fluorescent metal nanocluster; and (c)a second probe hybridized to the test nucleic acid, the second probecomprising (iv) an enhancer sequence that enhances fluorescence of thefluorescent metal nanocluster when the enhancer sequence interacts withthe nucleation sequence of the first probe; (v) an abasic site; and (vi)a hybridization sequence that is complementary to a second sequence inthe control nucleic acid sample.

A further embodiment concerns a kit comprising: (a) a control nucleicacid comprising a first and a second sequence, wherein said first andsecond sequence are separated by one nucleotide, which comprises anepigenetic modification; (b) a first probe hybridized to the testnucleic acid, the first probe comprising (i) a hybridization sequencethat is complementary to the first sequence in the control nucleic acid;(ii) an abasic site; and (iii) a nucleation sequence comprising afluorescent metal nanocluster; and (c) a second probe hybridized to thetest nucleic acid, the second probe comprising (iv) an enhancer sequencethat enhances fluorescence of the fluorescent metal nanocluster when theenhancer sequence interacts with the nucleation sequence of the firstprobe; (v) a recognition nucleotide that interacts with the nucleotidethat comprises the epigenetic modification in the control nucleic acid;and (vi) a hybridization sequence that is complementary to a secondsequence in the control nucleic acid sample.

As used herein, “essentially free,” in terms of a specified component,is used herein to mean that none of the specified component has beenpurposefully formulated into a composition and/or is present only as acontaminant or in trace amounts. The total amount of the specifiedcomponent resulting from any unintended contamination of a compositionis therefore well below 0.05%, preferably below 0.01%. Most preferred isa composition in which no amount of the specified component can bedetected with standard analytical methods.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising,” the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein. The patent or application filecontains at least one drawing executed in color. Copies of this patentor patent application publication with color drawing(s) will be providedby the Office upon request and payment of the necessary fee.

FIG. 1: N⁶-methyladenine detection using methyladenine-specificNanoCluster Beacons (maNCBs) (not drawn to scale). Consisting of an NCprobe (i.e. the cytosine-rich Ag cluster-nucleation sequence) and anenhancer probe (i.e. the guanine-rich sequence), maNCB forms two 3-wayjunctions (3WJ) when hybridizing with the two targets (A target or A*target; A* represents m6A). Here only the nucleotides-of-interest (A* orA) on the two targets and the recognition nucleotides on the two probes(n or x) are shown. The nomenclatures that represent the two resultingprobe-target hybridization complexes are nxA and nxA*, respectively. Thelowercase letters, n and x, indicate the recognition nucleotides on theNC and the enhancer probes, respectively. The capital letter representsthe type of adenine on the target. Here x, the abasic site, serves as anon-interacting, neutral site, while the silver cluster-enhanceralignment is only controlled by the interactions between the NC probe'srecognition nucleotide n and the nucleotides-of-interest on the targets.The maNCB design goal is thus to identify a suitable recognitionnucleotide for the NC probe that can generate two differentiable silvercluster emission spectra upon probe-target binding.

FIGS. 2A-2D: (A) 2D fluorescence contour plots of gxA and gxA* thatclearly differentiate between A* and A targets (indicated by thickarrows). The only difference between these two targets (60-nt long) isan A to A* (m6A) substitution right in the middle of the sequence(Target Set 1 in Table 1). Arrow pointing upwards indicates a strongerA-g interaction as compared to A*-g interaction. (B) Emission profilesof gxA and gxA* (intensity normalized at 670 nm) along the dashed linein A (with Stokes shift of 60 nm). Error bars (represented by ribbons)show standard deviations from five trials. The P value is found to be0.0001 at 645 nm. (C-D) When using t or a as recognition nucleotides(txA vs. txA* and axA vs. axA*), no significant changes in 2D spectraare observed. The target sequence used here has the GATC motif (TargetSet 1).

FIGS. 3A-3C: maNCBs containing two canonical recognition nucleotidesfailed to discriminate A* target from A target. 2D fluorescence contourplots of the selected recognition nucleotide combinations: (A) gtA vs.gtA*, (B) ttA vs. ttA* and (C) atA vs. atA*. None of these sets showsignificant changes in their emission spectra. Target sequence used herehas the GATC motif (Target Set 1).

FIGS. 4A-4B: (A) A-G base pairing via trans Hoogsteen/sugar edgeconfiguration is found in many RNA structures. In this purine-purinebase pairing, N⁶H of A and 2′OH of G are in close proximity to eachother (indicated by arrows), forming a hydrogen bond between them. (B)Presence of the bulky N⁶-methyl group of m⁶A (denoted as A*) hinders theabove H-bond formation and may sterically clash with the phosphatebackbone of G⁹ (indicated by arrows), causing A*-G base pair to beweaker than A-G pair.

FIG. 5: Quantification of adenine methylation extent in heterogeneoussamples using high-resolution melting analysis. High-resolution meltinganalysis were performed in the six dsDNA mixtures. Here fraction 0 means100% A target and fraction 1 means 100% A* target. Fraction 0 has higherintensity while fraction 1 has lower intensity. After normalization at75° C. and 83° C. (T_(m)±4° C.), intercalating dye's emission intensityat 79° C. is found correlated to the amount of A* target in the mixture(R²=0.899). Target sequence used here has the GATC motif (Target Set 1).The dsDNA used here contains either an A-g or an A*-g base pair in thesequence (different from the dsDNA used in FIG. 3B). The meltingtemperatures of fraction 0 and 1 samples indicate that the A-g pair ismore stable than the A*-g pair even in DNA.

FIGS. 6A-6D: (A) 2D fluorescence contour plots of gxA and gxA* thatclearly differentiate between A* and A targets (indicated by thickarrows). The only difference between these two targets (60-nt long) isan A to A* substitution right in the middle of the sequence (Target Set2 in Table 1). The arrow pointing upwards indicates a stronger A-ginteraction as compared to A*-g interaction. (B) Emission profiles ofgxA and gxA* (intensity normalized at 670 nm) along the dashed line in A(with Stokes shift of 60 nm). Error bars (represented by ribbons) showstandard deviations from five trials. The P value is found to be 0.0008at 645 nm. (C-D) When using t or a as recognition nucleotides (txA vs.txA* and axA vs. axA*), no significant changes in 2D spectra areobserved. Target sequence used here has the GATC motif (Target Set 2).

FIGS. 7A-7D: (A) 2D fluorescence contour plots of txA and txA* thatclearly differentiate between A* and A targets (indicated by thickarrows). The only difference between these two targets (60-nt long) isan A to A* substitution right in the middle of the sequence (Target Set3 in Table 2). The arrow pointing upwards indicates a stronger A-tinteraction as compared to A*-t interaction. (B) Emission profiles oftxA and txA* (intensity normalized at 670 nm) along the black dash linein A (with Stokes shift of 60 nm). Error bars (represented by ribbons)show standard deviations from five trials. The P value is found to be0.0002 at 620 nm. (C-D) When using g or a as recognition nucleotides(gxA vs. gxA* and axA vs. axA*), no significant changes in 2D spectraare observed. Target sequence used here has the CTGCAG motif (Target Set3).

FIGS. 8A-8D: (A) 2D fluorescence contour plots of txA and txA* thatclearly differentiate between A* and A targets (indicated by thickarrows). The only difference between these two targets (60-nt long) isan A to A* substitution right in the middle of the sequence (Target Set4 in Table 2). The arrow pointing upwards indicates a stronger A-tinteraction as compared to A*-t interaction. (B) Emission profiles oftxA and txA* (intensity normalized at 670 nm) along the dashed line in A(with Stokes shift of 60 nm). Error bars (represented by ribbons) showstandard deviations from five trials. The P value is found to be 0.0002at 620 nm. (C-D) When using g or a as recognition nucleotides (gxA vs.gxA* and axA vs. axA*), no significant changes in 2D spectra areobserved. Target sequence used here has the CTGCAG motif (Target Set 4).

FIGS. 9A-9B: Quantification of adenine methylation extent inheterogeneous samples. (A) Six samples with different A* targetfractions (Target Set 3) were prepared and mixed with maNCB_3. Herefraction 0 means 100% A target and fraction 1 means 100% A* target.Fraction 0 has higher intensity and fraction 1 has lower intensity.After normalization at 670 nm, maNCB's emission intensity at 620 nm isfound correlated to the amount of A* target in the mixture (R² range:0.922 to 0.976). (B) High-resolution melting analysis is also performedin the six dsDNA mixtures. After normalization at 71° C. and 79° C.(T_(m)±4° C.), intercalating dye's emission intensity at 75° C. is alsofound correlated to the amount of A* target in the mixture (R²=0.984).Target sequence used here has the CTGCAG motif (Target Set 3). The dsDNAused here contains either an A-t or an A*-t base pair in the sequence(Table 3). It was previously shown that A-T pair is more stable thanA*-T pair in DNA.

FIGS. 10A-10B: Quantification of adenine methylation extent inheterogeneous samples. (A) Six samples with different A* targetfractions (Target Set 1) were prepared and mixed with maNCB_1. Herefraction 0 means 100% A target and fraction 1 means 100% A* target.Fraction 0 has higher intensity and fraction 1 has lower intensity.After normalization at 670 nm, maNCB's emission intensity at 645 nm isfound correlated to the amount of A* target in the mixture (R2 range:0.817 to 0.951). (B) High-resolution melting analysis are also performedin the six dsDNA mixtures. After normalization at 75° C. and 83° C.(Tm±4° C.), intercalating dye's emission intensity at 79° C. was alsofound correlated to the amount of A* target in the mixture (R2=0.909).Target sequence used here has the GATC motif (Target Set 1). It waspreviously shown that A-T pair is more stable than A*-T pair in DNA(Lopez et al., 2012).

FIG. 11: maNCB can pinpoint the location of a specific m⁶A in the DNAsequence. maNCB_1 is designed to target the adenine within the GATCmotif (site 1) while maNCB_5 targets a nearby adenine (site 2). Emissionspectra from the unmethylated target-probe mixtures are used asbackground for subtraction. The background-subtracted spectrum ofmaNCB_5 clearly indicates that maNCB_5 cannot detect m⁶A within the GATCmotif, while the background-subtracted maNCB_1 spectrum shows anunambiguous detection result of m⁶A within the GATC motif at 645 nm(P˜0.002). Error bars (represented by ribbons) show standard deviationsfrom five trials.

FIG. 12: maNCB can pinpoint the location of a specific m⁶A in the DNAsequence. maNCB_3 is designed to target the adenine within the CTGCAGmotif (site 1) while maNCB_6 (Table 2) targets a nearby adenine (site2). Emission spectra from the unmethylated target-probe mixtures areused as background for subtraction. The background-subtracted spectrumof maNCB_6 clearly indicates that maNCB_6 cannot detect m⁶A within theCTGCAG motif, while the background-subtracted maNCB_3 spectrum shows anunambiguous detection result of m⁶A within the CTGCAG motif at 620 nm(P<0.001). Error bars (represented by ribbons) show standard deviationsfrom five trials.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Embodiments of the present invention provide compositions and methodsfor detecting and/or quantifying epigenetically modified nucleic acidmolecules. In particular, it has been found that by using a paired probeset including a metal nanocluster and a probe having an enhancersequence, epigenetically modified nucleotide positions can be detectedin the same reaction by a change in fluorescence signal from the metalnanocluster. In some aspects, probes for use according to theembodiments comprise two single-stranded nucleic acid probes that aredesigned to complement with contiguous sections of a target nucleicacid. One of the probes of the pair includes a recognition nucleotide(which interacts with a candidate site for epigenetic modification on atarget sequence) and a “nucleation portion” having a nucleotide sequencethat can bind to metal nanoclusters. The other probe of the pairincludes an “enhancer portion” that includes a nucleotide sequence whichcan produces an enhancement in fluorescence from the nanoclusters whenthe enhancer sequence is in sufficient proximity to the nanoclusters andthe hybridized probe is subjected to excitation light (e.g., ultravioletlight). When the two probes are brought into sufficient proximity, suchas by, binding the contiguous sections of the target nucleic acidsequence, the nucleation portion (and the metal nanoclusters) and theenhancement portion are in close proximity, which can be detected bydetecting an increase in fluorescence intensity of the metalnanoclusters. However, when the target nucleic acid includes anepigenetic modification that interacts with the recognition nucleotideof the probe (e.g., a N⁶-methyladenine) the alignment of the metalnanoclusters and the enhancer sequence is offset. Offsetting thisalignment causes a resulting change in the wavelength (e.g., thefluorescence spectra) of the fluorescence emitted from the nanoclusters,which can be detected, and used to identify the variant in the varianttarget nucleic acid sequence. Moreover, the fluorescence intensity canbe used to quantify the proportion of target nucleic acid molecules thatinclude the given epigenetic modification.

I. DEFINITIONS

The term “complementary binding” as used herein occurs when the base ofone nucleic acid molecule forms a hydrogen bond to the base of anothernucleic acid molecule. Normally, the base adenine (A) is complementaryto thymidine (T) and uracil (U), while cytosine (C) is complementary toguanine (G). For example, the sequence 5′-ATCG-3′ of one ssDNA moleculecan bond to 3′-TAGC-5′ of another ssDNA to form a dsDNA. In thisexample, the sequence 5′-ATCG-3′ is the reverse complement of3′-TAGC-5′. Nucleic acid molecules can be complementary to each othereven without complete hydrogen-bonding of all bases of each molecule.For example, hybridization with a complementary nucleic acid sequencecan occur under conditions of differing stringency in which a complementwill bind at some but not all nucleotide positions.

“Control” refers to a sample or standard used for comparison with anexperimental sample. In some embodiments, the control is a sampleobtained from a healthy subject (or a plurality of healthy subjects),such as a subject or subjects not expected or known to have a particularpolymorphism. In additional embodiments, the control is a historicalcontrol or standard reference value or range of values (such as apreviously tested control sample or plurality of such samples), or groupof samples that represent baseline or normal values. A positive controlcan be an established standard that is indicative of a specificmethylated nucleotide. In some embodiments a control nucleic acid is onethat lacks a particular methylated nucleotide, and is used in assays forcomparison with a test nucleic acid, to determine if the test nucleicacid includes the methylated nucleotide.

“Detecting” is used herein to identify the existence, presence, or factof something. General methods of detecting are known to the skilledartisan and may be supplemented with the protocols and reagentsdisclosed herein. For example, included herein are methods of detectinga nucleic acid molecule in sample. Detection can include a physicalreadout, such as fluorescence output.

“Enhancer Sequence” refers to a nucleotide sequence that when placed inproximity to another nucleic acid molecule having templated metalnanoclusters increases the fluorescence intensity of the metalnanocluster when exposed to excitation light. Exemplary enhancersequences are known in the art and disclosed herein.

“Excitation Light” refers to light of any wavelength that is capable ofcausing template metal nanoclusters to fluoresce. Non-limiting examplesof excitation light include visible light, ultraviolet and near infraredlight.

The term “hybridization” is defined as forming base pairs betweencomplementary regions of two strands of DNA, RNA, or between DNA andRNA, thereby forming a duplex molecule, for example. Hybridizationconditions resulting in particular degrees of stringency will varydepending upon the nature of the hybridization method and thecomposition and length of the hybridizing nucleic acid sequences.Generally, the temperature of hybridization and the ionic strength (suchas the Na+ concentration) of the hybridization buffer will determine thestringency of hybridization. Calculations regarding hybridizationconditions for attaining particular degrees of stringency are discussedin Sambrook et al., (1989) Molecular Cloning, second edition, ColdSpring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11).

An “isolated” biological component (such as a nucleic acid molecule) hasbeen substantially separated, produced apart from, or purified away fromother biological components. Nucleic acid molecules which have been“isolated” include nucleic acids molecules purified by standardpurification methods, as well as those chemically synthesized. Isolateddoes not require absolute purity, and can include nucleic acid moleculesthat are at least 50% isolated, such as at least 75%, 80%, 90%, 95%,98%, 99% or even 100% isolated.

“Metal nanoclusters” are collections of small numbers (e.g., 2-30 atoms)of noble metal atoms (e.g., gold or silver atoms) with physical sizesclose to the Fermi wavelength of an electron (^(˜)0.5 nm for gold andsilver). The metal atoms can have affinity for nitrogen atoms on DNA,including the N3 of cytosine and the N7 of guanine. Metal nanoclustersfor use with the disclosed embodiments are fluorescent, that is, theyhave the ability to emit light of a particular wavelength (emissionwavelength) when exposed to light of another wavelength (excitationwavelength).

A “nucleation sequence” is a sequence of nucleotides capable of bindingor associating with metal atoms to form template metal nanoclusters. Theportion of a nucleic acid molecule including a nucleation sequence ofnucleotides is referred to as the “nucleation portion” of the nucleicacid molecule. Exemplary nucleation sequences are known and providedherein. Specific nucleation sequences that are useful for interactingwith metal nanoclusters and forming DNA templated metal nanoclusters aredisclosed herein. Examples of metal nanoclusters for use as fluorescentreporters, and methods of producing templated metal nanoclusters on DNAoligonucleotides are known. See, e.g., U.S. Patent Publication No.US20110212540, incorporated by reference herein in its entirety, andU.S. Publication No. US20140349289, incorporated herein by reference.

A “nucleic acid” is a deoxyribonucleotide or ribonucleotide polymer,which can include analogues of natural nucleotides that hybridize tonucleic acid molecules in a manner similar to naturally occurringnucleotides. In a particular example, a nucleic acid molecule is asingle stranded (ss) DNA or RNA molecule, such as a probe or primer. Inanother particular example, a nucleic acid molecule is a double stranded(ds) nucleic acid, such as a target nucleic acid. Examples of modifiednucleic acids are those with altered backbones, such as peptide nucleicacids (PNA).

“Probes” are short nucleic acid molecules, usually DNA oligonucleotides,typically of about 20-100 nucleotides in length, used to detect thepresence of a complementary target DNA strand in a sample. All or aportion of a probe can be annealed to a complementary target DNA strandby nucleic acid hybridization to form a hybrid between the primer andthe target DNA strand. Therefore, probes can be used to identify atarget nucleic acid molecule, wherein the sequence of the probe isspecific for the target nucleic acid molecule, for example so that theprobe will hybridize to the target nucleic acid molecule under very highstringency hybridization conditions.

Typically, probes include at least about 10 contiguous nucleotides, suchas at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49 or about 50 contiguous nucleotides, thatare complementary to a target nucleic acid molecule, such as 20-70nucleotides, 20-60 nucleotides, 20-50 nucleotides, 20-40 nucleotides, or20-30 nucleotides. Probes can also be of a maximum length, for exampleno more than 20, 25, 25, 40, 50, 75 or 100 nucleotides in length. Thespecificity of a particular probe typically increases with an increasein the number of complementary nucleotides on the probe.

The probe can also include additional nucleotides that are notcomplementary to the target nucleic acid molecule. The additionalnucleotides can be used, for example, for detection of the probe in asample. In several embodiments, the probes disclosed herein include ahybridization portion that is complementary to a test nucleic acidsequence, and a nucleation portion (that can associate with metalnanoclusters) or an enhancer portion (that can enhance the fluorescenceof metal nanoclusters associated with the nucleation portion. Theadditional nucleotides can be located 5′ or 3′ of the hybridizationnucleotides.

Methods for preparing and using nucleic acid probes are described, forexample, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual,CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1998), and Innis et al.(PCR Protocols, A Guide to Methods and Applications, Academic Press,Inc., San Diego, Calif., 1990).

A “sample,” such as a biological sample, is a sample obtained from asubject. As used herein, biological samples include all clinical samplesuseful for detection of a methylated nucleotide, including, but notlimited to, cells, tissues, and bodily fluids, such as: blood;derivatives and fractions of blood, such as serum; urine; sputum; or CVSsamples. In a particular example, a sample includes blood obtained froma human subject, such as whole blood or serum.

A “test nucleic acid molecule” refers to a nucleic acid molecule whosedetection, quantitation, qualitative detection, characterization, or acombination thereof, is intended. For example, the test nucleic acidmolecule can be a defined region or particular portion of a nucleic acidmolecule, for example a portion of a genome (such as a gene or a regionof DNA or RNA containing a gene (or portion thereof) of interest). Thenucleic acid molecule need not be in a purified form. Various othernucleic acid molecules can also be present with the test nucleic acidmolecule. For example, the test nucleic acid molecule can be a specificnucleic acid molecule (which can include RNA or DNA), for which thedetection of a particular polymorphism is intended. In some examples, atest nucleic acid includes a viral nucleic acid molecule, or a bacterialnucleic acid molecule. Purification or isolation of the test nucleicacid molecule, if needed, can be conducted by methods known to those inthe art, such as by using a commercially available purification kit orthe like.

An “abasic” site or “AP (apurinic/apyrimidinic)” site is referred toherein as a location in nucleic acid (e.g., DNA or RNA) that has neithera purine nor a pyrimidine base. An abasic site may occur spontaneouslyor due to DNA damage. AP sites are one of the most frequent spontaneouslesions in DNA.

II. EPIGENETIC MODIFICATIONS

The number of nucleic acid modifications identified in the genomes andtranscriptomes has exploded since the first discovery of non-canonicalnucleobases six decades ago (Rozenski et al., 1999; McCloskey et al.,2005; Behm-Ansmant et al., 2011). Many of the modifications in genomesare heritable epigenetic marks that influence the way the genes areexpressed and eventually define cell status among the higher organisms(Song et al., 2012). In particular, N⁶-methyladenine (m6a) is amethylation modification abundant in prokaryotic genomes (Low et al.,2011), and also found in lower eukaryotes (Hattman 2005; Ratel et al.,2006; Wion et al., 2006) and higher plants (Vanyushin et al., 1988).While the biological functions of m6A at GATC sites in single-celledorganisms are well studied (such as genome defense, mismatch repair andgene expression control (Marinus et al., 1987)), its roles in eukaryoticgenomes remain largely unknown (Ratel et al., 2006).

So far detection of N⁶-methyladenine in DNA has been demonstrated usinga single-molecule, real-time sequencing method (Flusberg et al., 2010).Detection of N⁶-methyladenosine in RNA has been shown using (1) nucleasecleavage followed by thin-layer chromatography (TLC) or massspectrometry (MS) (Kellner et al., 2010), (2) immunocapturing ofm6A-containing RNA fragments followed by sequencing (Dominissini et al.,2013), (3) ligation (Dai et al., 2007), and (4) a selective polymerase(Harcourt et al., 2013). However, these methods are laborious (e.g.require multiple steps to identify a single site), time-consuming (e.g.TLC and MS), and high-cost (e.g. enzymatic reaction). Whereashigh-resolution melting (HRM) analysis is able to detect a single m6Amodification within a target DNA via the destabilizing effect of m6A,HRM cannot pinpoint the location of m6A in the sequence (Lopez et al.,2012). A simple and cost-effective way to identify single m6A at anyspecific sites is therefore highly desired.

The techniques detailed here provide robust, simple, enzyme-free andhybridization-based method for m6A detection with pinpoint specificity,using a new type of silver cluster-based DNA probe which are termedmethyladenine-specific NanoCluster Beacon (maNCB). Yeh and co-workershave previously introduced a NanoCluster Beacon (NCB) that fluorescesupon binding to a DNA target (Yeh et al., 2010; Yeh et al., 2011;Obliosca et al., 2014; Juul et al., 2015). NCBs employ DNA-templated,few-atom silver nanoclusters (DNA/Ag NCs, about 2-20 silver atoms percluster) as reporters whose fluorescence can be significantly enhancedthrough interactions with a nearby G-rich sequence (called an enhancer).Not only is the fluorescence of silver clusters activated, but thefluorescence color can also respond to the “alignment” of silver clusterwith respect to the enhancer sequence (Yeh et al., 2012). In otherwords, the fluorescence emission of silver clusters is sensitive to thenucleobase environment surrounding the clusters. Taking advantage ofthis fluorescence tunability by altering the surrounding ligands, aproperty that is not commonly seen among existing reporters (Oblisoscaet al., 2013), NCB soon evolved to a multicolor probe, termed chameleonNanoCluster Beacon (cNCB), for single-nucleotide polymorphism (SNP)detection (Yeh et al., 2012). Here we bring the NCB detection to thenext level by designing a new NCB specifically for m6A detection.

III. DETECTING METHYLATED NUCLEOTIDES

A method is disclosed for detecting a methylated nucleotide between atest nucleic acid and a control nucleic acid using metal nanoclusterbeacons (NCB). The NCB includes two probes that each include portionscomplementary to contiguous sections of the control nucleic acidsequence. The two probes can also include regions of complementaritywith each other, e.g., to make the three-way-junction (3WJ) discussedbelow. One of the probes of the NCB includes a “nucleation portion” thatincludes a nucleotide sequence that can bind to metal nanoclusters. Theother probe of the NCB includes an “enhancer portion” that includes anucleotide sequence that can enhance fluorescence emitted from thenanoclusters when in sufficient proximity to the nanoclusters. When thetwo probes are brought into sufficient proximity by binding thecontiguous sections of the control nucleic acid sequence, the nucleationportion (and the metal nanoclusters) and the enhancement portion are inclose proximity, which can be detected by detecting an increase influorescence intensity of the metal nanoclusters.

The first probe also includes a nucleation portion including templatedmetal nanoclusters, which can fluoresce when exposed to excitation light(e.g., UV light). The second probe includes an enhancer portionincluding a nucleotide sequence that enhances fluorescent emission fromthe metal nanoclusters when associated with the metal nanoclusters.

The test mixture is exposed to excitation light, and any correspondingfluorescence is measured. Detecting a difference in the wavelength ofthe fluorescence emission of the test mixture compared to that of acorresponding control mixture comprising the first probe, the secondprobe, and the control nucleic acid molecule detects the presence of amethylated nucleotide in the test nucleic acid molecule compared to thecontrol nucleic acid molecule. The difference in wavelength fluorescenceemission between the test mixture and the control mixture can be avisible color difference. In more embodiments, the difference inwavelength fluorescence emission between the test mixture and thecontrol mixture is at least 1 nm (such as at least 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 nm). The testnucleic acid molecule may be compared to multiple different referencestandard nucleic acid molecules.

In some embodiments, the methylated nucleotide is N⁶-methyladenine. Forexample, the probes hydridize around methylated or unmethylatedadenosine. The measured fluorescence of the control mixture serves as areference standard for comparison purposes to the measured fluorescenceof the test mixture. Where the control mixture and the test mixtureprovide the same or substantially similar measurable wavelength or rangeof wavelengths upon exposure to excitation light, it may be concludedthat the test nucleic acid molecule and the control nucleic acidmolecule have the same nucleotide sequence across the junction of thehybridization portions of the first and second probes. Where the controlmixture and the test mixture provide the different measurable wavelengthor range of wavelengths upon exposure to excitation light, it may beconcluded that the test nucleic acid molecule and the control nucleicacid molecule do not have the same nucleotide sequence across thejunction of the hybridization portions of the first and second probes.In certain aspects, the measured fluorescence between the controlmixture and test mixture provides quantification of theN⁶-methyladenine.

A. Metal Nanoclusters

The disclosed embodiments take advantage of the fluorescent propertiesof metal (e.g., silver) nanoclusters when the nanoclusters are broughtnear a DNA sequence referred to herein as an “enhancer sequence”. Themetal of the templated metal nanoclusters can be a noble metal, such assilver, gold, or copper. DNA-templated silver nanoclusters can emitcolored light through interactions with “enhancer” sequences. The colorof the emitted light was found to depend on the particular enhancersequence. Silver nanoclusters are groups of from about 2 to about 30silver atoms that are sub 2 nm in size with the properties of goodfluorescence, good photostability, and electroluminescence. These silvernanoclusters, which are templated on the nucleation sequence of anembodiment probe, function as fluorescence reporters.

To form metal nanoclusters on DNA, positively charged metal ions (e.g.,Ag+ atoms) are first attached to ssDNA (e.g., cytosine nucleotides)spontaneously in solution. Then, a reductant (e.g., sodium borohydride)is added to reduce the charge of the atoms (e.g., Ag+ to Ag(0)), afterwhich metal atom “clusters” will form. The ssDNA prevents the metalcluster “from growing out of control”. Clusters that become a“nanoparticle” (size >5 nm) are not fluorescent.

Examples of metal nanoclusters for use as fluorescent reporters, andmethods of producing templated metal nanoclusters on DNAoligonucleotides are known. See U.S. Pat. App. Pub. 2011/0212540,incorporated by reference herein in its entirety. The basis for theoperation of the templated metal nanoclusters is a controlled conversionof DNA-templated silver nanoclusters between a dark, non-emissive state,which is their state when not associated with an enhancer sequence, anda bright, emissive state when associated with the enhancer sequence.Unlike prior use of metal nanoclusters, the present method involvestuning the fluorescent emission properties of the metal nanoclusters(e.g., a wavelength shift of 60-70 nm) by altering the relativepositions of Noble metal nanoclusters, such as those made of silver,gold, copper, or other noble metals typically include collections of anumber of metal atoms (approximately 2-30 atoms or 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29 or 30 atoms) with physical sizes close to the Fermiwavelength of an electron (e.g., about 0.5 nm for gold and silver). Theybehave like molecular systems and yield fluorescence emission in theUV-visible and infrared range. In some examples,oligonucleotide-templated silver nanoclusters (“DNA/Ag NCs”), which area versatile set of fluorophores that have been used for a variety ofapplications including live cell imaging, detection of specific metalions, and single-nucleotide variation identification. DNA/Ag NCs can bebiocompatible and can have better photostability than commonly usedorganic dyes. Unlike organic dyes and photoluminescent nanocrystals,they are subject to silver oxidation/reduction or nanocluster (“NC”)regrouping, which results in conversion among different NC species.These different species may provide different color emissions.

In the disclosed embodiments, the proximity of metal nanoclusters withan enhancer sequence occurs when the NC and enhancer portions of twoprobes of the NCB are brought into close proximity by the hybridizationof the hybridization portion of the probes with a contiguous portion ofa target nucleic acid molecule. Prior to hybridization, the nanoclustersare only weakly fluorescent or non-fluorescent. After hybridization, thefluorescence emission from the silver nanoclusters is enhanced (theprobe “lights up”) because the nanoclusters which are templated onto thenucleation sequence are brought into proximity with the guanine(s) fromthe enhancer sequence. As discussed in more detail herein, by alteringthe alignment of the metal nanoclusters with the enhancer sequence, theemission wavelength of light emitted from the nanoclusters can bealtered.

The association of the metal nanoclusters on the first probe with theenhancement portion of the second probe can increase the fluorescenceemission of the templated metal nanoclusters by at least 2-fold fold. Ina related aspect, the association of the metal nanoclusters on the firstprobe with the enhancement portion of the second probe can increase thefluorescence emission of the templated metal nanoclusters from about2-fold to about 500-fold (such as about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475 or 500-fold).

Detection of fluorescence emission can be performed according to knownmethods, for example as described herein. The excitation light can beselected from the group consisting of ultraviolet light, visible light,near infrared light or a combination thereof. In a related aspect, thewavelength of excitation light is from 200 nm to 2000 nm (or 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575,600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925,950, 975, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950 or 2000 nm).

B. Nucleic Acid Samples

DNA and/or RNA samples may be obtained from a variety of sources, asfurther detailed herein. In certain cases, a plurality of nucleic acidsamples are analyzed in parallel. Samples for parallel analysis mayinclude a nucleic acid standard having a known level of methylation.Likewise, a plurality of different oligonucleotide probes may beemployed to determine methylation status at multiple regions within anucleic acid sample.

Exemplary eukaryotic nucleic acids that can be used in a method of theinvention includes, without limitation, mammal nucleic acids such as arodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig,goat, cow, cat, dog, primate, human or non-human primate. Plant DNA mayalso be analyzed according to the invention. For example, nucleic acidsfrom Arabidopsis thaliana, maize, sorghum, oat, wheat, rice, canola, orsoybean may be analyzed. It is further contemplated that nucleic fromother organisms such as algae, a nematodes, insects (e.g., Drosophilamelanogaster, mosquito, fruit fly, honey bee or spider), fish, reptiles,amphibians and yeast may be analyzed.

As indicated above, DNA and/or RNA can be isolated from one or morecells, bodily fluids or tissues. An array of methods can be used toisolate DNA and/or RNA from samples such as blood, sweat, tears, lymph,urine, saliva, semen, cerebrospinal fluid, feces or amniotic fluid.Genomic DNA can also be obtained from one or more cell or tissue inprimary culture, in a propagated cell line, a fixed archival sample,forensic sample or archeological sample. Methods for isolating genomicDNA and/or RNA from a cell, fluid or tissue are well known in the art(see, e.g., Sambrook et al., 2001).

Exemplary cell types from which nucleic acids can be obtained in amethod of the invention include, a blood cell such as a B lymphocyte, Tlymphocyte, leukocyte, erythrocyte, macrophage, or neutrophil; a musclecell such as a skeletal cell, smooth muscle cell or cardiac muscle cell;germ cell such as a sperm or egg; epithelial cell; connective tissuecell such as an adipocyte, fibroblast or osteoblast; neuron; astrocyte;stromal cell; kidney cell; pancreatic cell; liver cell; or keratinocyte.A cell from which DNA and/or RNA is obtained can be at a particulardevelopmental level including, for example, a hematopoietic stem cell ora cell that arises from a hematopoietic stem cell such as a red bloodcell, B lymphocyte, T lymphocyte, natural killer cell, neutrophil,basophil, eosinophil, monocyte, macrophage, or platelet. Other cellsinclude a bone marrow stromal cell (mesenchymal stem cell) or a cellthat develops therefrom such as a bone cell (osteocyte), cartilage cells(chondrocyte), fat cell (adipocyte), or other kinds of connective tissuecells such as one found in tendons; neural stem cell or a cell it givesrise to including, for example, a nerve cells (neuron), astrocyte oroligodendrocyte; epithelial stem cell or a cell that arises from anepithelial stem cell such as an absorptive cell, goblet cell, Panethcell, or enteroendocrine cell; skin stem cell; epidermal stem cell; orfollicular stem cell. Generally any type of stem cell can be usedincluding, without limitation, an embryonic stem cell, adult stem cell,totipotent stem cell or pluripotent stem cell.

A cell from which a nucleic acid sample is obtained for use in theinvention can be a normal cell or a cell displaying one or more symptomof a particular disease or condition. Thus, a nucleic acid sample usedin a method of the invention can be obtained from a cancer cell,neoplastic cell, apoptotic cell, senescent cell, necrotic cell, anautoimmune cell, a call comprising a heritable genetic disease or thelike.

Additionally, the test and control nucleic acids can be processed ormanipulated, such as being amplified, digested by restrictionendonucleases, or labeled. In some embodiments, the nucleic acids arediluted in one or more solutions, arranged in an array, and/or placed ona solid substrate (for example, a DNA microchip). In certainembodiments, the nucleic acids are diluted in an organic or inorganicsolvent to form solutions. The solution optionally can containadditives, such as stabilizers, preservatives, or buffers.

The target DNA samples may contain specific sequences for hybridizationto the probes. For example, for detection of m6A, targets may be GATCand/or CTGCAG-containing target samples.

IV. REAGENTS AND KITS

The kits may comprise suitably aliquoted reagents of the presentinvention, such as an enhancer probe and NC probe. Additional componentsthat may be included in a kit according to the invention include, butare not limited to, control oligonucleotides (e.g., methylated andnon-methylated oligonucleotides), distilled water, probes, dyes, samplevials and instructions for performing methylation quantification assays.In certain further aspects, reagents for DNA isolation, DNA purificationand/or DNA clean-up may also be included in a kit. In some examples, oneor more sets of probes, may be provided in pre-measured single useamounts in individual, typically disposable, tubes or equivalentcontainers. With such an arrangement, the sample to be tested for thepresence of the target nucleic acids can be added to the individualtube(s) and amplification carried out directly. In some embodiments,kits also may include the reagents necessary to carry out fluorescencedetection assays, including sample preparation reagents, appropriatebuffers, salts, tubes or assay cells.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there is more than one component in the kit, the kitalso will generally contain a second, third or other additionalcontainer into which the additional components may be separately placed.However, various combinations of components may be comprised in a vial.The kits of the present invention also will typically include a meansfor containing reagent containers in close confinement for commercialsale. Such containers may include cardboard containers or injection orblow-molded plastic containers into which the desired vials areretained.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being preferred. However, the components of the kit maybe provided as dried powder(s). When reagents and/or components areprovided as a dry powder, the powder can be reconstituted by theaddition of a suitable solvent. It is envisioned that the solvent mayalso be provided in another container means.

V. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Methyladenine-Specific NanoCluster Beacons for GATC-ContainingTargets

Similar to cNCB, maNCB adopts a binary probe configuration that forms a3-way junction (3WJ) with the DNA target (FIG. 1). Consisting of an NCprobe (i.e. the C-rich Ag cluster-nucleation sequence shown in blue) andan enhancer probe (i.e. the G-rich sequence shown in red), maNCB bindsto the target around the “nucleotide-of-interest,” which is either m6A(N6-methyladenine, denoted as A* in FIG. 1) or A (unmethylated adenine)in this study. Since the most common occurrence of m6A was identifiedwithin GATC sites (Marinus et al., 2009), two 60-nt long targetscontaining either a GATC or a GA*TC site were synthesized (Target Set 1in Table 1). These two targets have exactly identical sequences exceptfor a single-nucleotide substitution (A→A*) in the middle (denoted asthe “A target” and the “A* target” respectively in FIG. 1). Upon 3WJformation between the probe and the target, two “recognitionnucleotides” (one on the NC probe and the other on the enhancer probe)are brought close to the nucleotide-of-interest on the target. In theprevious SNP detection using cNCB, Watson-Crick basepairing is formedbetween the nucleotide-of-interest and one of the recognitionnucleotides, leading to two distinct alignment states between the silvercluster and the enhancer sequence. The main task in the maNCB design istherefore to search for a set of recognition nucleotides that candiscriminate m6A from A by distinct emission spectra of activated silverclusters.

TABLE 1 Oligonucleotide sequences of maNCBsused for Target Sets 1 and 2. Name DNA Sequence (5′→3′) Target Set 1Unmethylated target ATC AAG TAC AGA TCA TGC (SEQ ID NO. 1)GTT GCA CGG TCG ATC AAG TAC AGA TCA TGC GTC GGG CTC GGAMethylated target ATC AAG TAC AGA TCA TGC (SEQ ID NO. 2)GTT GCA CGG TCG A*TC AAG TAC AGA TCA TGC GTC GGG CTC GGA maNCB_1NC probe CCC TTA ATC CCC g CGA (SEQ ID NO. 3) CCG TGC AAC GCA TGA TCTGTA Enhancer probe CCC GAC GCA TGA TCT GTA (SEQ ID NO. 4)CTT GAx GGG TGG GGT GGG GTG GGG Control NC Probes_1 NC Probe_aCCC TTA ATC CCC a CGA (SEQ ID NO. 5) CCG TGC AAC GCA TGA TCT GTANC Probe_t CCC TTA ATC CCC t CGA (SEQ ID NO. 6) CCG TGC AAC GCA TGA TCTGTA maNCB_5 NC probe CCC TTA ATC CCC g ACT (SEQ ID NO. 7)TGA TCG ACC GTG CAA CGC ATG Enhancer probe TCC GAG CCC GAC GCA TGA(SEQ ID NO. 8) TCT Gx G GGT GGG GTG GGG TGG GG Target Set 2Unmethylated target CTA GTT TAA TTT TGT TTT (SEQ ID NO. 9)GTG GGT TAA AAG ATC GTT TAA ATC AAT ATT TAC AAC ATA AAAMethylated target CTA GTT TAA TTT TGT TTT (SEQ ID NO. 10)GTG GGT TAA AAG A*TC GTT TAA ATC AAT ATT TAC AAC ATA AAA maNCB_2NC probe CCC TTA ATC CCC g CTT (SEQ ID NO. 11) TTA ACC CAC AAA ACA AAATTA Enhancer probe GTT GTA AAT ATT GAT TTA (SEQ ID NO. 12)AAC GAx GGG TGG GGT GGG GTG GGG Control NC Probes_2 NC Probe_aCCC TTA ATC CCC a CTT (SEQ ID NO. 13) TTA ACC CAC AAA ACA AAA TTANC Probe_t CCC TTA ATC CCC t CTT (SEQ ID NO. 14) TTA ACC CAC AAA ACA AAATTA

Included are the unmethylated adenine (A) and N6-methyladenine (denotedas A*), the cytosine-rich nucleation sequence, the recognitionnucleotide, the hybridization sequence, the abasic site (x) and theG-rich enhancer sequence. Target 1 is a synthetic target (Flusberg etal., 2010) while Target 2 (Cooper et al., 2014) is related to a stx2ACDS gene (GenBank: CP006027.1; 3,056,718 . . . 3,056,777).

The inventors tested all recognition nucleotide combinations but failedto achieve a working maNCB design (FIG. 3). In the second attempt, thenumber of recognition nucleotides was reduced from two to one byintroducing an AP site to the enhancer probe (denoted as x in FIG. 1).Out of the four recognition nucleotides tested on the NC probe, only gcould give differentiable silver cluster emission spectra uponprobe-target binding (spectrum difference pointed by arrows in FIG. 2A).The difference in 2D spectra can be more clearly seen in 1D spectra(FIG. 2B), which were plotted along a 45° line (with a fixed Stokesshift of 60 nm) and normalized at 670 nm. The two 1D spectraunambiguously deviated from each other at 645 nm, with p value of about0.0001. On the other hand, when other recognition nucleotides were used,no differentiation was observed (FIGS. 2C and 2D). The discriminationgiven by g must be due to different interaction strengths between A-gand A*-g. Dai et al. have previously identified guanine to be the mosteffective recognition base in their ligation-based method forN6-methyladenosine detection on a RNA target (Dai et al., 2007). In RNA,adenosine can form a sheared, non-Watson-Crick base pair with guanosine,in which three hydrogen bonds are established guanosine (FIG. 4)(Leontis et al., 2002). In the crowded space between the Hoogsteen andsugar edges (Walczak), a bulky N6-methyl group can (1) hinder the H-bondformation between N6H of adenosine and 2′OH of guanosine and (2) causesteric clash with the phosphate backbone of guanosine. In this case,while the former has no contribution since both the targets and probesare DNA, the latter can weaken A*-g interaction that results in thedifferentiation seen in FIG. 2. The dsDNA with an A*-g pair in themiddle had a melting temperature lower than that of the dsDNA with anA-g pair (FIG. 5). While testing the same maNCB design (using g as therecognition nucleotide for NC probe) on a second GATC-containing targetset29 (Target Set 2 in Table 1), differentiable spectra, similar tothose from Target Set 1, were obtained (FIG. 6).

Example 2 Methyladenine-Specific NanoCluster Beacons forCTGCAG-Containing Targets

Whereas the first maNCB design worked well for the GATC site, it couldnot differentiate m6A from A on the CTGCAG site (Target Sets 3 & 4 inTable 2). Instead, t served as a good recognition nucleotide for NCprobe to detect m6A on the CTGCAG sites (FIGS. 7 and 8). Upon closerexamination, the resulting spectra from the second maNCB design (where tis used as recognition nucleotide) were very different from thoseobtained from the first maNCB design (where g is used as recognitionnucleotide). The gxA and gxA* complexes (from Target Sets 1 & 2) led totwo major fluorescent species which emitted at 645 nm and 670 nm,respectively (FIGS. 2B and 6B). Here the differentiation of m6A from Awas actually caused by suppression of the 645 nm species relative to the670 nm species in the gxA* complex. On the other hand, the txA and txA*complexes (from Target Sets 3 & 4) produced at least three major specieswhich emitted at 620 nm, 645 nm, and 670 nm (FIGS. 7B and 8B). The m6Adifferentiation here was caused by suppression of the 620 nm speciesrelative to the 670 nm species in the txA* complex. While it is notclear why there were two major fluorescent species in one case (withGATC site) and three species in another case (with CTGCAG site), it isobvious that subtle changes in the ligand environment favor theformation of one cluster species over another, enabling the m6Adetection through a simple “two-color analysis”. In the maNCB detection,the 670 nm emission served as the normalization standard (FIGS. 2B, 6B,7B and 8B) while the relative intensity at 645 nm (Target Sets 1 & 2) or620 nm (Target Sets 3 & 4) could differentiate m6A from A. Thus, thesemaNCB detection results were highly reproducible and consistent amongtargets (P<<0.05 in FIGS. 2, 6, 7 and 8).

TABLE 2 Oligonucleotide sequences of maNCBsused for Target Sets 3 and 4. Name DNA Sequence (5′→3′) Target Set 3Unmethylated target CAA TAC ATT ATG GGA AAG (SEQ ID NO. 15)TAA TAC AGC TGC AGC GTT TCT GAA CAG AAA GTC ACA GTT TTTMethylated target CAA TAC ATT ATG GGA AAG (SEQ ID NO. 16)TAA TAC AGC TGC A*GC GTT TCT GAA CAG AAA GTC ACA GTT TTT maNCB_3NC probe CCC TTA ATC CCC t GCA (SEQ ID NO. 17)  GCT GTA TTA CTT TCC CATAAT Enhancer probe TGT GAC TTT CTG TTC AGA (SEQ ID NO. 18)AAC GCx GGG TGG GGT GGG GTG GGG Control NC Probes_3 NC probe_aCCC TTA ATC CCC a GCA (SEQ ID NO. 19) GCT GTA TTA CTT TCC CAT AATNC probe_g CCC TTA ATC CCC g GCA (SEQ ID NO. 20) GCT GTA TTA CTT TCC CATAAT mNCB_6 NC probe CCC TTA ATC CCC t GTA (SEQ ID NO. 21)TTA CTT TCC CAT AAT GTA TTG Enhancer probe TTT CTG TTC AGA AAC GCT(SEQ ID NO. 22) GCA GCx GGG TGG GGT GGG GTG GGG Target Set 4Unmethylated target ATC TTG TAA CGC AGC ATC (SEQ ID NO. 23)CAG ACG TCC TGC AGC CAG ATC GGA ATA GAC CAA ATC CTG GTTMethylated target ATC TTG TAA CGC AGC ATC (SEQ ID No. 24)CAG ACG TCC TGC A*GC CAG ATC GGA ATA GAC CAA ATC CTG GTT maNCB_4NC probe CCC TTA ATC CCC t GCA (SEQ ID NO. 25) GGA CGT CTG GAT GCT GCGTTA Enhancer probe GAT TTG GTC TAT TCC GAT (SEQ ID NO. 26)CTG GCx GGG TGG GGT GGG GTG GGG Control NC Probes_4 NC probe_aCCC TTA ATC CCC a GCA (SEQ ID NO. 27) GGA CGT CTG GAT GCT GCG TTANC probe_g CCC TTA ATC CCC g GCA (SEQ ID NO. 28) GGA CGT CTG GAT GCT GCGTTA The unmethylated adenine (A) and N⁶-methyladenine (denoted as A*),the cytosine-rich nucleation sequence, the recognition nucleotide, thehybridization sequence, the abasic site (x) and the G-rich enhancersequence are shown. Target 3 (Hemday et al., 2002) is related to a papBpromoter gene (GenBank: M63747.1; 282..341) while Target 4 (Fang et al.,2013) is associated to a lysine/arginine/ornithine transporter subunitCDS (GenBank: CP010344.1; 2,526,243..2,526,302).

TABLE 3 Oligonucleotide Sequences used forhigh-resolution melting analysis. Name DNA Sequence (5′→3′)Target Set 1 complementary strand Target Set 1_gCCC GAC GCA TGA TCT GTA CTT GAg (SEQ ID NO. 29)CGA CCG TGC AAC GCA TGA TCT GTA Target Set 1_tCCC GAC GCA TGA TCT GTA CTT GAt (SEQ ID NO. 30)CGA CCG TGC AAC GCA TGA TCT GTA Target Set 3 complementary strandTarget Set 3_t TGT GAC TTT CTG TTC AGA AAC GCt (SEQ ID NO. 31)GCA GCT GTA TTA CTT TCC CAT AAT

Example 3 Alternate Nucleation Sequences

The nucleation sequence of the nucleation probe can be altered tovarious lengths. For example, the number of cytosines in the nucleationsequence maNCB were varied in the nucleation probe (Table 4) and foundto function similarly. Thus, the number of cytosines can generally bevaried to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 cytosines.

TABLE 4 Nucleation sequences with various number of cytosines. NameDNA Sequence (5′→3′) NC probe CC TTA ATC C g CGA CCG (SEQ ID NO. 32)TGC AAC GCA TGA TCT GTA NC probe CCC TTA ATC CC g CGA CCG(SEQ ID NO. 33) TGC AAC GCA TGA TCT GTA NC probeCCCC TTA ATC CCC g CGA CCG (SEQ ID NO. 34) TGC AAC GCA TGA TCT GTANC probe CCCCC TTA ATC CCCC g CGA CCG (SEQ ID NO. 35)TGC AAC GCA TGA TCT GTA NC probe CCCCCC TTA ATC CCCCC g CGA CCG(SEQ ID NO. 36) TGC AAC GCA TGA TCT GTA NC probeCCCCCCC TTA ATC CCCCCC g CGA CCG (SEQ ID NO. 37) TGC AAC GCA TGA TCT GTANC probe CCCCCCCC TTA ATC CCCCCCC g CGA CCG (SEQ ID NO. 38)TGC AAC GCA TGA TCT GTA

Example 4 maNCB Quantification and Pinpoint Specificity

Taking advantage of the large relative-intensity change at 620/645 nm,maNCB can precisely quantify the degree of methylation in heterogeneoussamples. Nucleic acid targets are not always fully modified, whereincomplete modification can be due to low availability of theco-substrates and limited modification enzyme activities (Saikia et al.,2006). To evaluate maNCB quantification in heterogeneous samples, the A*and A targets were mixed at six different ratios and compared theresulting relative intensities at a fixed wavelength (645 nm for TargetSet 1 in FIG. 10A and 620 nm for Target Set 3 in FIG. 9A). As expected,maNCB's relative emission at 645 nm was found proportional to the A*target fraction, demonstrating the quantification of methylation degreein heterogeneous samples by maNCB (FIG. 10A). As shown in FIGS. 10B and9B (see Table 3 for sequences), high-resolution melting (HRM) analysiscan also be used for m6A detection (Lopez et al., 20120. Whereasnormalized fluorescence intensity of intercalating dye (EvaGreen) alsoshowed a linear trend with A* amount and could be used to quantify theA*/A mixing ratio, the melting analysis cannot “pinpoint” the locationof a specific A* modification on the target.

To demonstrate the capability of maNCB to pinpoint the location of aspecific m6A, two maNCBs, which target different adenines in the sametarget, were designed and tested. As expected, the background-subtractedspectra (FIG. 11) clearly indicated that m6A at site 1 can only bedetected by maNCB_1 (P<<0.05 at 645 nm), but not maNCB_5 (Table 1). This“pinpoint specificity” in m6A detection was also preserved in thetargets with CTGCAG site (FIG. 12 and Table 2).

In summary, the inventors demonstrated the use of maNCBs for m6Adetection at the single-base resolution. This enzyme-free detectionmethod comes with high reproducibility and can directly quantify theextent of adenine modification at a particular site in heterogeneoussamples. To date, there is no hybridization technique that has thepotential to reach these remarkable results. It is expected that theconcept of maNCB can be generally applied to the detection of differenttypes of methylation modifications such as 5-methylcytosine (Herman etal., 1996), N4-methylcytosine (Ehrlich et al., 1985),5-hydroxymethylcytosine (Pfeifer et al., 2013) and N7-methylguanine(Rottman et al., 1974), as long as the suitable recognition nucleotidesare identified in the NCB design.

Example 5 Materials and Methods

Sodium phosphate dibasic anhydrous (Na₂HPO₄; F.W. 141.96), sodiumphosphate monobasic monohydrate (NaH₂PO₄.H₂O; F.W. 137.99), zinc acetateand sodium borohydride (NaBH₄) were purchased from Fisher Scientific,Precision Melt Supermix was obtained from Bio-Rad, whereas silvernitrate (AgNO₃) was acquired from Sigma-Aldrich. HPLC purifiedmethylated and unmethylated DNA targets were synthesized by TriLinkBiotechnologies (San Diego, Calif., USA). All other oligonucleotideswere purchased from Integrated DNA Technologies (Coralville, Iowa, USA)and were purified by desalting. De-ionized (DI) water (18 MΩ-cm) wasused for all solution preparations.

Preparation of Methyladenine-Specific NanoCluster Beacons (maNCBs)Preparation of Nanocluster Probes (NC Probes)

In a typical preparation, a 15 μM (final concentration) nanoclusterprobe (NC probe) solution with a volume of 1 ml was prepared by adding12.5 μl of 1.2 mM NC probe to 940 μl of 20 mM sodium phosphate buffer(pH 6.7). The solution was vortexed for 2 s and centrifuged at 14,000rpm for 30 s. 45 μl of 4 mM silver nitrate solution was then added andthe mixture was again vortexed and centrifuged. The solution was allowedto sit in the dark for 10 min at room temperature. For silver clusterformation, 7 μl of freshly prepared 13.2 μM NaBH₄ solution was added,resulting in a pale yellow mixture which was then stored in the darkovernight. The resulting NC probe solution had the NC probe:Ag⁺:NaBH₄molar ratio of 1:12:6.

Activation of maNCB

Consisting of an NC probe (i.e. the cytosine-rich Ag cluster-nucleationsequence originally carrying dark silver clusters) and an enhancer probe(i.e. the guanine-rich sequence), maNCB employs a 3-way junctionhybridization configuration with a target. When maNCB binds to a target,the enhancer probe is brought close to the NC probe throughhybridization and the dark silver clusters are activated and becomehighly emissive through the interactions with the nearby guanine-richenhancer sequence. We call this process “the guanine-proximity-inducedactivation of silver clusters” or, in short, “the activation of maNCBs”.To activate maNCBs, 13.3 μl of 0.5 mM zinc acetate was added to a 120 μlaliquot of 10 μM NC probe solution (concentration adjusted from thepreviously prepared 15 μM NC probe) and the resulting mixture wasvortexed. 1 μl of 1.2 mM of an enhancer probe and of a target were addedto this mixture, vortexed and immersed in a hot water bath (90-95° C.)for 1 min, followed by gradual cooling to room temperature for 45 min.The activated maNCB had the NC probe:enhancer probe:target molar ratioof 1:1:1.

Fluorescence Measurements

Fluorescence emission and excitation scans were performed on aFluoroMax-4 spectrofluorometer from Horiba Scientific. 120 μl NCB samplewas placed in a 100 μl quartz cuvette (16.100F-Q-10/Z15, Starna Cells)for fluorometer measurements. For the 2D measurement, the excitation andemission were scanned from 200 to 800 nm and 400 nm to 800 nm,respectively using 5 nm slit size, 5 nm increment step, and 0.1 sintegration time.

High-Resolution Melting Analysis

Single-stranded DNAs were added to the Precision Melt Supermix®(Bio-Rad, USA) to obtain a final DNA concentration of 10 μM. Allmeltings were performed in 12 μl volumes. The melting curve analysis wasconducted on CFX Connect Real-Time System (CFX Manager software version1.6, Bio-Rad, USA) using the SYBR channel. Before melting, samples weredenatured at 93° C. for 1 min and cooled down to room temperature for 30min. During melting, the temperature was increased from 30 to 100° C. in0.2° C. incremental steps, with each step held for 2 s. For meltinganalysis, the relative fluorescence units (RFU) of the resulting meltingcurves were first plotted against melting temperatures (Tm).Normalization regions were selected before and after the major decreasein fluorescence4 (Tm±4° C.). Comparisons between methylated andunmethylated DNA (fraction analysis) were made in terms of Tm (79° C.for Target Set 1 and 75° C. for Target Set 3).

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method for detecting an epigenetic modification in a test nucleicacid sample comprising: (I) hybridizing the test nucleic acid sample toa first and second probe, wherein (a) the first probe comprises (i) ahybridization sequence that is complementary to a first sequence in thetest nucleic acid sample; (ii) an abasic site or a recognitionnucleotide that interacts with the nucleotide that comprises theepigenetic modification in the test nucleic acid; and (iii) a nucleationsequence comprising a fluorescent metal nanocluster; and (b) the secondprobe comprises (iv) an enhancer sequence that enhances fluorescence ofthe fluorescent metal nanocluster when the enhancer sequence interactswith the nucleation sequence of the first probe; (v) an abasic site or arecognition nucleotide that interacts with the nucleotide that comprisesthe epigenetic modification in the test nucleic acid; and (vi) ahybridization sequence that is complementary to a second sequence in thetest nucleic acid sample; wherein if the first probe comprises arecognition nucleotide then the second probe comprises an abasic site;wherein if the first probe comprises an abasic site then the secondprobe comprises a recognition nucleotide; (II) exposing the test nucleicacid sample and the first and second probes to an excitation light; and(III) detecting a fluorescence signal from the fluorescent metalnanocluster, thereby detecting an epigenetic modification in the testnucleic acid sample.
 2. The method of claim 1, wherein step (I)comprises hybridizing the test nucleic acid sample to a first and secondprobe, wherein (a) the first probe comprises (i) a hybridizationsequence that is complementary to a first sequence in the test nucleicacid sample; (ii) a recognition nucleotide that interacts with thenucleotide that comprises the epigenetic modification in the testnucleic acid; and (iii) a nucleation sequence comprising a fluorescentmetal nanocluster; and (b) the second probe comprises (iv) an enhancersequence that enhances fluorescence of the fluorescent metal nanoclusterwhen the enhancer sequence interacts with the nucleation sequence of thefirst probe; (v) an abasic site; and (vi) a hybridization sequence thatis complementary to a second sequence in the test nucleic acid sample.3. The method of claim 1, wherein step (I) comprises hybridizing thetest nucleic acid sample to a first and second probe, wherein (a) thefirst probe comprises (i) a hybridization sequence that is complementaryto a first sequence in the test nucleic acid sample; (ii) an abasicsite; and (iii) a nucleation sequence comprising a fluorescent metalnanocluster; and (b) the second probe comprises (iv) an enhancersequence that enhances fluorescence of the fluorescent metal nanoclusterwhen the enhancer sequence interacts with the nucleation sequence of thefirst probe; (v) a recognition nucleotide that interacts with thenucleotide that comprises the epigenetic modification in the testnucleic acid; and (vi) a hybridization sequence that is complementary toa second sequence in the test nucleic acid sample.
 4. The method ofclaim 1, wherein the first sequence in the nucleic acid sample ispositioned 5′ relative to the second sequence of the nucleic acidsample.
 5. The method of claim 1, wherein the first sequence in thenucleic acid sample and the second sequence of the nucleic acid sampleare separated by one nucleotide, which is the candidate nucleotide. 6.The method of claim 1, wherein (III) detecting the fluorescence signalfrom the fluorescent metal nanocluster comprises detecting a change inthe fluorescence signal detected in the test nucleic acid sample ascompared to a control nucleic acid sample.
 7. The method of claim 6,wherein the change in the fluorescence signal is a change in thewavelength of the fluorescence signal.
 8. The method of claim 7, whereinthe change in the fluorescence signal is a change of at least 1 nm inthe wavelength of the fluorescence signal.
 9. The method of claim 6,wherein the control nucleic acid sample comprises nucleic acid moleculesthat are known to include the epigenetic modification.
 10. The method ofclaim 6, wherein the control nucleic acid sample comprises nucleic acidmolecules that are known not to include the epigenetic modification. 11.The method of claim 1, wherein detecting the fluorescence signal fromthe fluorescent metal nanocluster further comprises quantifying thefluorescence signal from the fluorescent metal nanocluster, therebyquantifying the proportion of nucleic acid molecules in the sample thatcomprise the epigenetic modification.
 12. The method of claim 11,wherein quantifying the fluorescence signal from the fluorescent metalnanocluster comprises quantifying a change in the fluorescence signaldetected in the test nucleic acid sample as compared to a controlnucleic acid sample.
 13. The method of claim 1, wherein the epigeneticmodification is selected from the group consisting of 5-methylcytosine,N⁴-methylcytosine, 5-hydroxymethylcytosine, N⁷-methylguanosine andN⁶-methyladenine.
 14. The method of claim 1, wherein the epigeneticmodification is N⁶-methyladenine. 15.-38. (canceled)
 39. A compositioncomprising: (a) a test nucleic acid comprising a first and a secondsequence, wherein said first and second sequence are separated by onenucleotide, which comprises an epigenetic modification; (b) a firstprobe hybridized to the test nucleic acid, the first probe comprising(i) a hybridization sequence that is complementary to the first sequencein the test nucleic acid; (ii) an abasic site or a recognitionnucleotide that interacts with the nucleotide that comprises theepigenetic modification in the test nucleic acid; and (iii) a nucleationsequence comprising a fluorescent metal nanocluster; and (c) a secondprobe hybridized to the test nucleic acid, the second probe comprising(iv) an enhancer sequence that enhances fluorescence of the fluorescentmetal nanocluster when the enhancer sequence interacts with thenucleation sequence of the first probe; (v) an abasic site or arecognition nucleotide that interacts with the nucleotide that comprisesthe epigenetic modification in the test nucleic acid; and (vi) ahybridization sequence that is complementary to a second sequence in thetest nucleic acid sample; wherein if the first probe comprises arecognition nucleotide then the second probe comprises an abasic site;wherein if the first probe comprises an abasic site then the secondprobe comprises a recognition nucleotide.
 40. The composition of claim39, wherein the first probe comprises (i) a hybridization sequence thatis complementary to the first sequence in the test nucleic acid; (ii) arecognition nucleotide that interacts with the nucleotide that comprisesthe epigenetic modification in the test nucleic acid; and (iii) anucleation sequence comprising a fluorescent metal nanocluster; and (c)a second probe hybridized to the test nucleic acid, the second probecomprising (iv) an enhancer sequence that enhances fluorescence of thefluorescent metal nanocluster when the enhancer sequence interacts withthe nucleation sequence of the first probe; (v) an abasic site; and (vi)a hybridization sequence that is complementary to a second sequence inthe test nucleic acid sample.
 41. The composition of claim 39, whereinthe first probe comprises (i) a hybridization sequence that iscomplementary to the first sequence in the test nucleic acid; (ii) anabasic site; and (iii) a nucleation sequence comprising a fluorescentmetal nanocluster; and (c) a second probe hybridized to the test nucleicacid, the second probe comprising (iv) an enhancer sequence thatenhances fluorescence of the fluorescent metal nanocluster when theenhancer sequence interacts with the nucleation sequence of the firstprobe; (v) a recognition nucleotide that interacts with the nucleotidethat comprises the epigenetic modification in the test nucleic acid; and(vi) a hybridization sequence that is complementary to a second sequencein the test nucleic acid sample.
 42. A kit comprising: (a) a controlnucleic acid comprising a first and a second sequence, wherein saidfirst and second sequence are separated by one nucleotide, whichcomprises an epigenetic modification; (b) a first probe hybridized tothe test nucleic acid, the first probe comprising (i) a hybridizationsequence that is complementary to the first sequence in the controlnucleic acid; (ii) an abasic site or recognition nucleotide thatinteracts with the nucleotide that comprises the epigenetic modificationin the control nucleic acid; and (iii) a nucleation sequence comprisinga fluorescent metal nanocluster; and (c) a second probe hybridized tothe test nucleic acid, the second probe comprising (iv) an enhancersequence that enhances fluorescence of the fluorescent metal nanoclusterwhen the enhancer sequence interacts with the nucleation sequence of thefirst probe; (v) an abasic site or a recognition nucleotide thatinteracts with the nucleotide that comprises the epigenetic modificationin the control nucleic acid; and (vi) a hybridization sequence that iscomplementary to a second sequence in the control nucleic acid sample;wherein if the first probe comprises a recognition nucleotide then thesecond probe comprises an abasic site; wherein if the first probecomprises an abasic site then the second probe comprises a recognitionnucleotide.
 43. The kit of claim 42, wherein the first probe comprises(i) a hybridization sequence that is complementary to the first sequencein the test nucleic acid; (ii) a recognition nucleotide that interactswith the nucleotide that comprises the epigenetic modification in thetest nucleic acid; and (iii) a nucleation sequence comprising afluorescent metal nanocluster; and the second probe comprises (iv) anenhancer sequence that enhances fluorescence of the fluorescent metalnanocluster when the enhancer sequence interacts with the nucleationsequence of the first probe; (v) an abasic site; and (vi) ahybridization sequence that is complementary to a second sequence in thetest nucleic acid sample.
 44. The kit of claim 42, wherein the firstprobe comprises (i) a hybridization sequence that is complementary tothe first sequence in the test nucleic acid; (ii) an abasic site; and(iii) a nucleation sequence comprising a fluorescent metal nanocluster;and the second probe comprises (iv) an enhancer sequence that enhancesfluorescence of the fluorescent metal nanocluster when the enhancersequence interacts with the nucleation sequence of the first probe; (v)a recognition nucleotide that interacts with the nucleotide thatcomprises the epigenetic modification in the test nucleic acid; and (vi)a hybridization sequence that is complementary to a second sequence inthe test nucleic acid sample.